Assays for measuring nucleic acid binding proteins and enzyme activities

ABSTRACT

The present invention provides processes for measuring DNA or RNA binding proteins, specific nucleic acids, as well as enzyme activities using labeled nucleic acids of labeled protein/peptide molecules.

FIELD OF INVENTION

The present invention describes processes for measuring DNA or RNAbinding proteins, specific nucleic acids, as well as enzyme activitiesusing labeled nucleic acids or labeled protein/peptide molecules.

BACKGROUND OF INVENTION

The ability to measure the activity or amount of an analyte in abiological sample is critical in the fields of life sciences researchand medical diagnostics. A broad class of important assays are assaysthat measure the activity of enzymes that catalyze the synthesis orcleavage of polypeptides or polynucleotides (or, similarly, assays forsubstrates, products or inhibitors of these enzymes). These enzymesinclude proteases, nucleases, polymerases, ligases and the like. Anotherbroad class of important assays measure the interaction of nucleic acidswith proteins or other nucleic acids.

Enzymatic activity may be measured through the use of synthetic enzymesubstrates that show changes in color or fluorescence when acted upon bythe enzyme. This approach, however, requires the design and synthesis ofa custom reagent for every enzyme; a process that can be laborious, timeconsuming, and expensive. In addition, it is often desirable to measurethe activity of an enzyme on its natural substrate.

A possibly more generic approach for measuring protease or nucleaseactivity is the Scintillation Proximity Assay (SPA); see, e.g., U.S.Pat. No. 4,568,649 and Published PCT Application WO90/03844. SPA usessmall microspheres that are derivatized in such a way as to bindspecific molecules. If a radioactive molecule is brought into closeproximity to the bead a scintillant incorporated in the microsphere isexcited and subsequently emits light. Radioactive molecules not bound tothe microspheres excite the scintillant to a much lesser extent thanradioactive molecules bound to the beads and, therefore, produce aweaker light signal. A number of assay formats have been described usingSPA detection technology including protease (Wilkinson et al., Pharm.Res. (1993) 10, 562) and ribonuclease protection assays (Kenrick, etal., Nucl. Acids Res. (1997) 25, 2947).

While SPA has proved useful for these and other classes of assays, thetechnique has several disadvantages. The primary problem with SPA is therequirement for radioactive reagents. Because of the severe cost,safety, environmental, and regulatory issues associated with the use ofradioisotopes, there is a clear need for alternative assay techniquesthat do not use radioactive materials. The background signal associatedwith the SPA approach is relatively high due to the inability of theassay format to totally discriminate the signal that is generated fromfree from that generated from bound radioactivity. In addition, thesensitivity of SPA has been found to be limited; there is a need formore sensitive assay techniques. As a result of the high backgroundsignal and low to moderate sensitivity, SPA approaches generally possessrelatively low signal to noise ratios which, in many cases, canadversely effect assay performance.

The most common method for measuring the specific interaction ofproteins with nucleic acids is the gel shift or electrophoretic mobilityshift assay. This approach has been widely used for the study ofsequence-specific binding proteins, especially transcription factors.The basis for the approach is that complexes of DNA and protein have areduced or “shifted” mobility during non-denaturing gel electrophoresis.DNA duplexes, containing a specific protein binding sequence, are endlabeled (generally with a radioactive label) and incubated with a samplecontaining the specific binding protein. The sample is subsequentlyanalyzed by electrophoresis and the specific complexes are detectedfollowing autoradiographic analysis of exposed film. The amount ofspecific binding protein is determined semi-quantitatively by measuringthe amount of the specific protein-DNA complex. This approach has beenlargely relegated to the world of basic exploratory research, primarilybecause of the inherent limitations of gel electrophoresis: i) thetechnique is complex and can usually only be carried out by highlytrained lab technicians; ii) the technique is slow and laborious and is,therefore, not suited to the high throughput screening of large numbersof samples; and iii) the technique is, at best, semi-quantitative innature. In addition, the use of radioactivity has also posed as anobstacle to some for the use of this technique. Although non-radioactiveapproaches have recently emerged, these approaches are accompanied bysignificant increases in labor.

Electrochemiluminescent Detection Technology

Numerous methods and systems have been developed for the detection andquantitation of molecules of interest in biochemical and biologicalsamples. Methods and systems which are capable of measuring traceamounts of microorganisms, pharmaceuticals, hormones, viruses,antibodies, nucleic acids and other proteins are of great value toresearchers and clinicians.

A very substantial body of art has been developed based upon bindingreactions, e.g., antigen-antibody reactions, nucleic acid hybridizationtechniques, protein-ligand systems as well as for formats for measuringa variety of enzymatic activities. The high degree of specificity inmany biochemical and biological assay systems has led to many methodsand systems of value in research and diagnostics. Typically, theexistence of an analyte or enzyme of interest is indicated by thepresence or absence of an observable “label” attached to one or more ofthe binding molecules or starting substrates.

Electrochemiluminescent (ECL) assays provide a sensitive and precisemeasurement of the presence and concentration of an analyte of interest.Such techniques use labels or other reactants that can be induced toluminesce when electrochemically oxidized or reduced in an appropriatechemical environment. Such electrochemiluminescence is triggered by avoltage impressed on a working electrode at a particular time and in aparticular manner. The light produced by the label is measured andindicates the presence or quantity of the analyte. For a fullerdescription of such ECL techniques, reference is made to U.S. Pat. No.5,714,089, U.S. Pat. No. 5,591,581, U.S. Pat. No. 5,597,910, U.S. Pat.No. 5,679,519, PCT published application WO90/05296, PCT publishedapplication WO92/14139, PCT published application WO90/05301; PCTpublished application WO96/24690, PCT published application U.S. Pat.No. 95/03190, PCT published application WO96/06946, PCT publishedapplication WO96/33411, PCT published application WO87/06706, PCTpublished application WO96/39534, PCT published application WO93/10267,PCT published application WO96/41175, PCT published applicationWO98/12539, PCT published application WO96/28538, PCT publishedapplication WO96/21039, PCT published application WO97/33176, PCTpublished application WO96/17248, and PCT published applicationWO96/40978, and U.S. patent application. Ser. No. 09/023,483. Thedisclosures of the aforesaid applications are hereby incorporated byreference in their entirety. Reference is also made to two reviews onECL technology: Blackburn et al. (Clinical Chemistry, 1991, 37,1534-1539) and a 1994 review of the analytical applications of ECL byKnight, et al. (Analyst, 1994, 119: 879-890) and the references citedtherein. The disclosure of the aforesaid articles are hereby alsoincorporated by reference in their entirety.

OBJECTS OF THE INVENTION

It is an object of the invention to provide a simple, accurate andreliable assay for measuring enzyme activity in a sample.

It is also an object of the invention to provide a simple, accurate andreliable assay for measuring proteins that bind nucleic acid.

It is an object of the invention to provide a simple, accurate andreliable assay for measuring inhibitors of enzyme activity in a sample.

It is an object of the invention to provide a simple, accurate andreliable assay for measuring substrates of enzymes in a sample.

It is an object of the invention to provide a simple, accurate andreliable assay for measuring specific nucleic acid sequences in asample.

SUMMARY OF THE INVENTION

The present invention describes processes for measuring DNA or RNAbinding proteins, specific nucleic acids, as well as enzyme activitiesusing labeled nucleic acids or labeled protein/peptide molecules. Asused herein, the term “measuring” or “measure” means detecting and/orquantitating.

This invention includes a method for measuring the amount or activity ofan enzyme in a sample that catalyzes the cleavage of a molecule into twoor more products, the method comprising the following steps: i) mixing asample which may contain the enzyme with a substrate of the enzyme, anECL label, and a solid phase, wherein the substrate is linked to the ECLlabel and is linked or capable of being linked to the solid phase andwherein the enzyme is capable of cleaving the substrate to form at leastone product that is linked to an ECL label but that is not linked orcapable of being linked to the solid phase; ii) inducing the mixture toemit electrochemiluminescence and iii) measuring theelectrochemiluminescence so as to measure the amount or activity of theenzyme. The invention also includes analogous methods for measuring theamount or activity of substrates or inhibitors of enzymes that catalyzethe cleavage of an enzyme into two or more products.

This invention includes a method for measuring the amount or activity ofan enzyme that catalyzes the joining of two or more substrates to form aproduct, the method comprising the following steps: i) mixing a samplewhich may contain the enzyme with two substrates of the enzyme, an ECLlabel, and a solid phase, wherein one of the substrates is linked orcapable of being linked to the solid phase and another of the substratesis not linked or capable of being linked to the solid phase but islinked to the ECL label and wherein the enzyme is capable of forming aproduct that is linked to the ECL label and linked or capable of beinglinked to the solid phase; ii) inducing the mixture to emitelectrochemiluminescence and iii) measuring the electrochemiluminescenceso as to measure the activity or amount of the enzyme. The inventionalso includes analogous methods for measuring the amount or activity ofsubstrates or inhibitors of enzymes that catalyze the joining of two ormore substrates to form a product.

This invention includes a method for measuring enzyme activities thatcleave nucleic acid molecules in a sample, which comprises, mixing atleast one or more single- or double-stranded nucleic acid moleculescontaining one or more ECL labels, adding a sample which may contain anucleic acid-cleaving enzyme, and incubating under conditions whichallow cleavage of the nucleic acid sequence, contacting this mixturewith at least one solid phase, preferentially inducing ECL from ECLlabels in solution or on the solid phase and measuring the ECL emissionso as to measure the amount of cleaving activity in the sample. Thisinvention includes a method for detecting and/or quantitating enzymeactivities that cleave peptide or protein molecules in a sample, whichcomprises, mixing at least one or more peptide or protein moleculescontaining one or more ECL labels, contacting this mixture with at leastone solid phase, adding a sample which may contain a peptide- orprotein-cleaving enzyme, inducing ECL from ECL labels in solution and/oron the solid phase and measuring the ECL emission so as to measure theamount of cleaving activity in the sample.

This invention includes a method for measuring enzyme activities thatcovalently join nucleic acid molecules in a sample, which comprises,mixing at least one or more single- or double-stranded nucleic acidmolecules containing one or more ECL labels, adding a sample which maycontain a nucleic acid-joining enzyme, incubating under conditions whichallow the joining of the nucleic acid sequences, contacting this mixturewith at least one solid phase, inducing ECL from ECL labels in solutionand/or on the solid phase and measuring the ECL emission so as tomeasure the amount of joining activity in the sample.

This invention includes a method for measuring enzyme activities thatcovalently join nucleic acid molecules in a sample, which comprises,mixing at least one or more single- or double-stranded nucleic acidmolecules containing one or more ECL labels, contacting one or more ofthe labeled nucleic acid molecules with at least one solid phase, addinga sample which may contain a nucleic acid-joining enzyme, incubatingunder conditions which allow the joining of the nucleic acid sequences,inducing ECL from ECL labels in solution and/or on the solid phase andmeasuring the ECL emission so as to measure the amount of joiningactivity in the sample.

This invention includes a method for measuring nucleic acid bindingproteins in a sample, which comprises the following steps: i) contactingthe sample with one or more single- and/or double-stranded nucleic acidmolecules containing a specific protein binding nucleotide sequence; ii)incubating under conditions which allow the specific binding of thenucleic acid binding proteins to the protein binding nucleotidesequence; iii) adding a nucleic acid cleaving reagent or enzyme; iv)incubating the sample under conditions that allow for the cleavage ofthe nucleic acid; and v) measuring the extent of nucleic acid cleavage.

This invention includes a method for detecting and/or quantitatingnucleic acid binding proteins in a sample, which comprises, mixing atleast one or more single- or double-stranded nucleic acid moleculescontaining a specific protein binding nucleotide sequence and containingone or more labels, contacting one or more of the labeled nucleic acidmolecules with at least one solid phase, adding a sample which maycontain one or more nucleic acid binding proteins, and incubating underconditions which allow the specific binding of the proteins to theprotein binding nucleotide sequence, adding a nucleic acid cleavingreagent or enzyme, and incubating the sample under conditions that allowfor the cleavage of the nucleic acid molecules, and measuring the amountof labeled nucleic acid on the solid phase and/or in the solution phase.

This invention includes a method for measuring nucleic acid bindingproteins in a sample, which comprises the following steps: i) contactingthe sample with one or more single- and/or double-stranded nucleic acidmolecules containing a specific protein binding nucleotide sequence andcontaining a number of modified nucleotides that are resistant tonuclease digestion; ii) incubating under conditions which allow thespecific binding of the proteins to the protein binding nucleotidesequence; iii) adding a nucleic acid cleaving reagent or enzyme; iv)incubating the sample under conditions that allow for the cleavage ofthe nucleic acid molecules; v) and measuring the extent of nucleic acidcleavage.

This invention includes a method for measuring nucleic acid bindingproteins in a sample, which comprises the following steps: i) mixing atleast one or more single- or double-stranded nucleic acid moleculescontaining a specific protein binding nucleotide sequence and containinga number of modified nucleotides that are resistant to nucleasedigestion, and containing one or more labels; ii) contacting one or moreof the labeled nucleic acid molecules with at least one solid phase;iii)adding a sample which may contain one or more nucleic acid bindingproteins, and incubating under conditions which allow the specificbinding of the proteins to the labeled nucleic acid sequences; iv)adding a nucleic acid cleaving reagent or enzyme, and incubating thesample under conditions that allow for the cleavage of the nucleic acidmolecules, and measuring the amount of label on the solid phase and/orin solution.

This invention includes a method for measuring nucleic acid bindingproteins in a sample, which comprises the following steps: mixing atleast one or more single- or double-stranded nucleic acid moleculescontaining a specific protein binding nucleotide sequence, andcontaining a number of modified nucleotides that are resistant tonuclease digestion, and containing one or more labels; adding a samplewhich may contain one or more nucleic acid binding proteins, andincubating under conditions which allow the specific binding of theproteins to the labeled nucleic acid sequences; adding a nucleic acidcleaving reagent or enzyme, and incubating the sample under conditionsthat allow for the cleavage of the nucleic acid molecules; contactingone or more of the labeled nucleic acid molecules with at least onesolid; and measuring the amount of labeled nucleic acid on the solidphase and/or in solution.

This invention includes a method for measuring nucleic acid bindingproteins in a sample, which comprises the following steps: mixing atleast one or more single- or double-stranded nucleic acid moleculescontaining a specific protein binding nucleotide, and containing anumber of modified nucleotides that are resistant to nuclease digestion,and containing one or more labels; contacting one or more of the labelednucleic acid molecules with at least one solid phase; adding a samplewhich may contain one or more nucleic acid binding proteins, andincubating under conditions which allow the specific binding of theproteins to the labeled nucleic acid sequences; adding a nucleic acidcleaving reagent or enzyme, and incubating the sample under conditionsthat allow for the cleavage of the nucleic acid molecules; and measuringthe amount of labeled nucleic acid on the solid phase and/or insolution.

This invention includes a method for measuring specific nucleic acidsequences in a sample, which comprises, mixing at least one or morepredetermined single-stranded nucleic acid molecules containing one ormore ECL labels, contacting one or more of the labeled nucleic acidmolecules with at least one solid phase, adding a sample which maycontain one or more of the specific nucleic acid sequences, incubatingunder conditions which allow the specific binding of the sample nucleicacid sequences to the labeled nucleic acid sequences, adding a nucleicacid cleaving reagent or enzyme, incubating the sample under conditionsthat allow for the cleavage of the nucleic acid molecules, and detectingand/or quantitating the amount of labeled nucleic acid on the solidphase and/or in solution.

This invention also includes a method for measuring specific nucleicacid sequences in a sample, which comprises, mixing at least one or morepredetermined single-stranded nucleic acid and containing one or moreECL labels, adding a sample which may contain one or more of thespecific nucleic acid sequences, incubating under conditions which allowthe specific binding of the sample nucleic acid sequences to the labelednucleic acid sequences, adding a nucleic acid cleaving reagent orenzyme, incubating the sample under conditions that allow for thecleavage of the nucleic acid molecules, contacting one or more of thelabeled nucleic acid molecules with at least one solid phase, measuringthe amount of labeled nucleic acid on the solid phase and/or insolution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows, schematically, an enzyme cleaving a substrate, which islinked to a solid phase and a label, to form a first product linked tothe label and a second product linked to the solid phase.

FIG. 2 shows, schematically, an enzyme joining a substrate linked to asolid phase with a substrate linked to a label, thereby, forming aproduct linked to both the label and solid phase.

FIGS. 3(a), (b) and (c) show, schematically, three methods by which asubstrate linked to a label and a binding reagent A can be contactedwith an cleaving enzyme and a binding reagent B (on a solid phase) so asto form a first product linked to the label and a second product linkedto the solid phase (by an A:B linkage).

FIGS. 4(a), (b) and (c) show, schematically, three methods by which afirst substrate, linked to a binding reagent A, and a second substrate,linked to a label, can be contacted with a joining enzyme and a bindingreagent B (on a solid phase) so as to form a product linked to both thelabel and the solid phase (by an A:B linkage).

FIGS. 5(a), (b), (c), (d), (e), and (f) illustrate six differentembodiments of the invention for measuring the activity of cleavingenzymes.

FIGS. 6(a), (b), (c), (d) and (e) illustrate five different embodimentsof the invention for measuring the activity of joining enzymes.

FIG. 7 illustrates a method for measuring the interaction of a nucleicacid nucleic acid binding protein.

FIG. 8 illustrates a method for measuring the activity of nucleic acidcleaving enzyme.

FIG. 9 the ECL signal measured in an ECL-based protease assay as afunction of the concentration of proteinase K.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention relates to assays for enzymes that cleave asubstrate into two or more products and/or join two or more substratesto form a product. The invention also includes assays for substrates andinhibitors of such enzymes. The enzyme activity is typically measured bythe ability of the enzyme to modulate the ECL signal generated from anECL label attached to a substrate molecule. The invention also includesreagents and kits for carrying out the methods of the invention. A kitfor carrying out the methods of the invention can comprise, in one ormore containers, at least two of the following components: enzyme,substrate, solid phase, buffers appropriate for carrying out theenzymatic reaction (e.g., mixtures of pH buffering substances,detergents, salts, metal ions, cofactors, proteins, sugars, excipients,and the like), solutions appropriate for carrying out an ECLmeasurement, solutions appropriate for cleaning and/or conditioning anECL measuring device, ECL labels, calibration solutions containing knownconcentrations of an enzyme, calibration solutions containing a knownconcentration of an enzyme inhibitor, and calibration solutions forcalibrating the response of an ECL measuring instrument. Suitablecontainers for such a kit include, but are not limited to vials,bottles, boxes, tubes blister packs, cartridges, syringes, microtiterplates, ampules, and the like.

As illustrated in FIG. 1, an enzyme 101 that catalyzes a cleavagereaction can be assayed through the use of a substrate 102 that islinked to both a solid phase 103 and an ECL label 104, so that theenzyme acts to separate the ECL label from the solid phase. The activityof the enzyme can be measured by an ECL measurement of the reduction ofthe number of ECL labels on the solid phase and/or an ECL measurement ofthe increase in the number of ECL labels in solution. In a differentembodiment, illustrated in FIG. 2, an enzyme 201 that catalyzes thejoining of two or more substrates is assayed through the use of asubstrate 202 that is linked to a solid phase 204 and another substrate203 that is linked to an ECL label 205, so that the enzyme acts to jointhe two substrates and, therefore, link the ECL label to the solidphase. The activity of the enzyme can be measured by an ECL measurementof the increase of the number of ECL labels on the solid phase and/or anECL measurement of the decrease in the number of ECL labels in solution.

The Solid Phase and Linking of Substrates

The term “solid phase” is understood to encompass a wide variety ofmaterials including solids, semi-solids, gels, films, membranes, meshes,felts, composites, particles, and the like. The solid phase can benon-porous or porous. Suitable solid phases include those developedand/or used as solid phases in solid phase binding assays (see, e.g.,chapter 9 of Immunoassay, E. P. Diamandis and T. K. Christopoulos eds.,Academic Press: New York, 1996, hereby incorporated by reference).

Methods for linking substrate molecules (e.g., polypeptides,polynucleotides, and polysaccharides) to solid phases are well known andinclude methods used for immobilizing reagents on solid phases for solidphase binding assays or for affinity chromatography (see, e.g.,Diamandis and Christopoulos cited above, and Hermanson, Greg T.,Immobilized Affinity Ligand Techniques, Academic Press: San Diego, 1992,hereby incorporated by reference). These methods include thenon-specific adsorption of molecules on the reagents on the solid phaseas well as the formation of a covalent bond between the reagent and thesolid phase. Alternatively, a substrate can be linked to a solid phasethrough a specific interaction with a binding group present on the solidphase (e.g., an antibody against a peptide substrate or a nucleic acidcomplementary to a sequence present on a nucleic acid substrate). In anadvantageous embodiment, a substrate or product labeled with a bindingreagent A (also referred to as a capture moiety) is contacted with asecond binding reagent B present on the surface of a solid phase, so asto link the substrate to the solid phase through an A:B linkage. Thisapproach is illustrated in FIGS. 3a-c (for assays of the activity ofsubstrate cleaving enzymes) and FIGS. 4a-c (for assays of the activityof substrate joining enzymes). FIGS. 3a-c show that a substrate 302,linked to both a label 304 and a binding reagent A, can be contactedwith a cleaving enzyme 301 prior to the formation of the A:B linkage(FIG. 3a), during the formation of the A:B linkage (FIG. 3b), oralternatively, after the formation of the A:B linkage (FIG. 3c) linkingbinding reagent A with a second binding reagent B present on the surfaceof the solid phase 303. Analogously, FIGS. 4a-c show that substrate 402,linked to a binding reagent A, and substrate 403, linked to a label 405,can be contacted with a joining enzyme 401 prior to the formation of theA:B linkage (FIG. 4a), during the formation of the A:B linkage (FIG.4b), or alternatively, after the formation of the A:B linkage (FIG. 4c)linking binding reagent A with a second binding reagent B present on thesurface of the solid phase 404. Many examples of A:B pairs that can beused to link molecules to a solid phase are known in the art, e.g.,antibody-hapten pairs, receptor-ligand pairs, complementary nucleic acidpairs, metal-metal ligand pairs, etc. In an especially advantageousembodiment, the A:B linkage is a biotin streptavidin interaction.

Solid phases particularly useful for ECL binding assays have beendescribed previously. One class of advantageous solid phases for ECLassays are particles. See published PC applications WO90/0530,WO89/04302, WO92/14138 and WO96/15440, said applications herebyincorporated by reference, for a description of particle-based reagents,methodology, and instrumentation for carrying out ECL binding assaysusing particles as a solid phase. These reagents, methodology andinstrumentation can be adapted for use in ECL-based enzyme assaysaccording to this invention. In an especially advantageous embodiment,magnetic particles (e.g., magnetic particles sold by Dynal, Seradyne, orImmunicon) are used as the solid phase. These particles can be collectedat an ECL working electrode through the application of a magnetic fieldso as to preferentially cause the excitation of ECL from ECL labelslinked to the particle, relative to ECL labels that are free insolution. Thus the amount of label on the solid phase can be measured inthe presence of free label, labeled substrate, or labeled product insolution. Labels linked to magnetic particles can be induced to emit ECLpreferentially (relative to labels in solution) by using a magneticfield to hold the particles on the electrode while free labels insolution are washed away. Alternatively, the labels in solution can bemeasured preferentially (relative to labels on magnetic particles) bycapturing the magnetic particles on a magnet distant from the electrodeused to induce ECL. There are several commercial instruments availablethat are capable of measuring the ECL emitted from magnetic particlescollected, through the use of a magnetic field, at an electrode (e.g.,ORIGEN, IGEN International; Elecsys, Boehringer-Mannheim; PicoLumi,Easai).

In an alternative embodiment, the solid phase is or comprises anelectrode for ECL measurements. The use of such solid phase forECL-based binding assays is described in PCT published applicationWO98/12539, hereby incorporated by reference in its entirety. The solidphases, instrumentation and methodology described in the above mentionedapplication can be adapted for use in the ECL-based enzyme assays of thepresent invention. Likewise, the methodology described in the abovementioned application for linking binding reagents to ECL electrodes canbe applied to the substrates and products of the current application.Suitable solid phases include metal electrodes (e.g., gold andplatinum), carbon electrodes (e.g., electrodes comprising glassy carbon,carbon black, graphite, carbon fibers, carbon nanotubes, and/or packedbeds of carbon fibers or nanotubes), and organic polymer-basedelectrodes. An especially advantageous solid phase comprises a compositeof carbon nanotubes in a polymer, e.g., a commercial plastic. Substratesand/or products can be linked to these and other solid phases by knownmethods including direct adsorption, covalent coupling, attachment to afilm or coating on the solid phase (e.g., molecules can be immobilizedon gold electrodes by attachment to a film of thiols coordinated to thegold surface, or silicon electrodes by attachment to a film of silanescovalently attached to silanols on the silicon surface), or by theformation of a specific A:B linkage (as was previously described in thepresent application). Advantageously, ECL labels in proximity to anelectrode surface (e.g., labels linked to enzyme substrates or productspresent on the surface of the electrode) can be preferentially inducedto emit ECL, relative to ECL labels that are free in solution. Thus theamount of label on the solid phase can be measured in the presence offree label, labeled substrate, or labeled product in solution. ECLlabels in proximity to an electrode surface can even more preferentiallybe induced to emit ECL (relative to ECL labels in solution) by washingaway the unbound label prior to inducing an ECL response. Alternatively,a solid phase distant from the electrode can be used to preferentiallyinduce ECL from labels not on the solid phase.

Substrates can be immobilized on different regions of one or more solidphases to form a patterned array of substrates. Such a patterned arrayhaving two or more regions comprising substrates that differ instructure from each other could be used to simultaneously measure theactivity of two or more enzymes (the substrates are chosen for theirknown specificity for a particular enzyme of interest). A similarpatterned array of a library of substrates can be used to determine thesubstrate specificity of a particular enzyme. By the application ofsolutions containing an enzyme and an inhibitor to defined regions on apatterned array of substrates, large numbers of inhibitors can berapidly screened for inhibitory ability. The measurement of the ECL froma patterned array can be conducted by imaging with an array of lightdetectors so that the ECL signal from different regions can bedistinguished and independently measured. Alternatively, the substratescan be patterned on an array of independent electrodes so that labels ina particular region can be selectively induced to emit ECL by theselective application of voltage to selected electrodes. In thisalternative embodiment, imaging is not necessary.

Ecl Labels

An ECL label is a chemical substance that, when electrochemicallyoxidized or reduced under appropriate conditions, emits light. The term“ECL label” or “electrochemiluminescent label” refers to the substanceitself, to a chemical derivative that has been modified to allowattachment to substrate or other reagent, or to a chemical derivativethat is attached to a substrate or other reagent. The term “ECL label”also refers to the various products and/or intermediates formed from thelabel during the ECL-generating reaction. Numerous ECL labels have beenreported in the literature (see the review by Knight et al., Analyst,119, 879, 1994). Useful ECL labels include polyaromatichydrocarbons(e.g., 9,10-diphenylanthracene, rubrene, phenanthrene,pyrene, and sulfonated derivatives thereof), organometallic complexes(e.g., complexes containing lanthanides, ruthenium, osmium, rhenium,platinum, chromium, and/or palladium), organic laser dyes, andchemiluminescent species (e.g., diacyl hydrazides such as luminol,acridinium esters, luiferase, and lucigenin). The ECL signal can beadvantageously increased by using labels comprising a polymer orparticle platform linked to a plurality of individual ECL labels (see,e.g., U.S. Pat. No. 5,679,519). Advantageous ECL labels are luminol andpolypyridyl (especially bipyridyl or phenanthrolyl)-containing complexesof ruthenium, osmium or rhenium (see, e.g., the complexes described inU.S. Pat. No. 5,714,089, U.S. Pat. No. 5,591,581, U.S. Pat. No.5,597,910, and published PCT application WO87/06706. The mostadvantageous ECL labels are ruthenium tris-bipyridyl (RuBpy) and itsderivatives. The term “RuBpy” refers to the substance itself, to achemical deri attachment to substrate or other reagent substrate (e.g.,derivatives comprising one or more of the following substituents: alkylgroups, amines, carboxylic acids, active esters or other activatedcarboxylic acid derivatives, phosphoramidites, hydrazides, alcohols,α,β-unsaturated carbonyls, aldehydes, ketones, halides or other leavinggroups, thiols, disulfides and the like), or to a chemical derivativethat is attached to a substrate or other reagent. The term “RuBpy” alsorefers to the various products and/or intermediates formed from thelabel during the ECL-generating reaction. RuBpy includes rutheniumtris-bipyridyl coupled to a variety of different types of biomoleculesto form highly ECL active conjugates. The ECL generated by oxidizing orreducing an ECL label at an electrode is known, for many ECL labels, tobe dramatically increased by the addition of another species (the ECLcoreactant) that is also oxidized or reduced at the electrode(generally, to give a reactive species that participates in a highlyenergetic reaction with the oxidation or reduction product of the ECLlabel). For example, hydrogen peroxide acts as an ECL coreactant forluminol. There are several classes of useful ECL coreactants for RuBpy,its derivatives, and the analogous osmium complexes, including:persulfate, oxalate, pyruvate (and other α-keto carboxylic acids), andamines (especially, tertiary amines). An advantageous method forinducing ECL from RuBpy-containing substances in biological assays isoxidation of the ECL label in the presence of a solution containingtripropylamine.

The Enzymes

The term “enzyme” is understood to cover all polypeptides (or analogsthereof) with catalytic activity (including naturally occurring enzymes,genetically modified enzymes, chemically modified enzymes, catalyticantibodies, enzyme fragments and synthetic polypeptides), as well asnucleic acids (or analogs thereof) with catalytic activity (includingribosomes, ribosomal RNA, and ribozymes), synthetic enzyme models ormimics (for example, synthetic molecules designed to mimic the catalyticsite of an enzyme), and enzyme cofactors that retain catalytic activityin the absence of a protein component. The enzymes that can be measuredby this technique include enzymes that cleave a substrate molecule intotwo or more products and/or enzymes that join two or more substratesinto a product. The enzymes can have both joining and cleaving activity,as in the following examples: i) a cleaving or joining enzyme that alsocatalyzes the reverse reaction (e.g., under appropriate conditions,proteases such as trypsin can catalyze the formation of amide bonds) andii) the enzyme (e.g., a transferase) catalyzes the transfer of a moietyfrom a first substrate (i.e., cleaving the moiety from the firstsubstrate) to a second substrate (i.e., joining the moiety to the secondsubstrate). Advantageously, the enzymes break or form a covalent bond.Especially advantageous enzymes are enzymes that break or form acovalent bond type from the following list: amide bond, ester bond,phosphodiester bond (e.g., the bond linking nucleotides in apolynucleotide), and disulfide bond. Some examples of classes of enzymesthat can be measured include the following: amidases, proteases,peptidases, glycosidases, saccharases, glycopeptidases, nucleases(including ribonucleases and deoxyribonucleases), endonucleases(including restriction endonucleases), exonucleases, ribosomes,ribosomal RNA, ribozymes, self-splicing molecules such as introns orinteins, esterases, phosphodiesterases, phosphorylases, APendonucleases, polymerases (e.g., DNA or RNA polymerases), nucleic acidrepair proteins, amino peptidases, carboxy peptidases, aminoacyl-tRNAsynthetases, ADP-ribosyl transferases, proteases of the complementpathway, proteases of the thrombolytic pathways, transferases,endoglycosidases, exoglycosidases, lipases, endoproteinases, glutathioneS-transferases, polysaccharide or oligosaccharide synthases (e.g.glycosyl transferases or glycogen synthases), ligases, ubiquitin-proteinligases, trans-glutaminases, integrases, and DNA glycosylases (e.g.,uracil-DNA glycosylases). The invention is not limited to enzymes thatcatalyze the formation or cleavage of covalent bonds; the invention alsocovers enzymes that catalyze the formation or cleavage of non-covalentbonds or binding interactions, e.g., enzymes that catalyze thehybridization or dehybridization of nucleic acids (RecA and the like),proteins that catalyze or promote the association of proteins (e.g.,Factor Va), peptides, and/or nucleic acids to form a complex, andproteins that catalyze the association of the peptide subunits of aprotein to form a protein. The enzyme can catalyze a reaction thatindirectly leads to the joining or cleavage of substrate molecules,e.g., the enzyme can catalyze a change in a substrate (e.g.,phosphorylation of a protein) that induces the substrate to bind to asecond molecule (e.g., a receptor specific for the phosphorylatedsubstrate). Similarly, the enzyme could catalyze a change in a substrate(e.g., phosphorylation of a protein or modification of a nucleic acid,that could target the substrate for degradation by a second enzyme orreagent). In another such embodiment, the enzyme converts a substratefrom an inactive form to a catalytically active form capable of cleavingor joining activity (e.g., Factor Xa can be measured from its ability toconvert the inactive prothrombin into the catalytically active cleavingenzyme thrombin, which is in turn measured through its ability to cleavea peptide substrate). The invention is not only limited to measuring theactivity of joining or cleaving enzymes but can also be used to measurethe activity of other reagents with similar activity, e.g., metalcomplexes or oxidizing agents, photoactive cleaving or crosslinkingagents, alkylating agents, acids, bases and the like that, that cleavebonds in nucleic acids, proteins, or polysaccharides (see, e.g., thefollowing publications, hereby incorporated by reference: Grant et al.Biochemistry, 1996, 35, 12313-12319; U.S. Pat. No. 4,980,473; andBiochemistry, G. Zubay, Ed., Addison-Wesley: Massachusetts, 1983).

The Substrates

The “substrates” that can be measured by the invention includesubstrates that are cleaved by an enzyme into two or more productsand/or substrates that are joined by an enzyme into a product.Advantageously, the substrates comprise polypeptides, polysaccharides,nucleic acids, amino acids, nucleotides, and/or sugars. The termspolypeptides and polysaccharides are understood to encompass,respectively, oligopeptides and oligonucleotides. The term nucleic acidsis understood to encompass oligonucleotides and polynucleotides. Thesubstrates can comprise analogs of polypeptides, polysaccharides,nucleic acids, amino acids, nucleotides, and/or sugars, e.g., i)polypeptides comprising unnatural amino acids, N-methyl amide linkages,and/or non-amide linkages; ii) nucleic acid analogs comprisingnon-phosphodiester linkages e.g., amide bonds (i.e., peptide nucleicacids—PNAs), phosphorothioate linkages, and/or methyl phosphonatelinkages; or iii) polysaccharides comprising non-glycosidic linkages(e.g., thioglycosides). In some embodiments, the substrates can bepolypeptides, polynucleotides, or polysaccharides that comprise bothnatural and unatural monomer units; the catalytic activity of cleavingor joining enzymes can by this method be restricted to the naturalcomponents of the substrate. The number of unnatural monomer units in asubstrate is advantageously between 1 and 999. In assays of the activityof enzymes that take modified nucleic acids or proteins as substrates(e.g., proteases specific for phosphorylated proteins or peptides,protesases specific for ubiquitinated proteins or peptides, DNA repairenzymes, etc.), the substrates can include the modified sites recognizedby the enzymes (e.g., phosphorylated amino acids, ubiquinated aminoacids, methylated nucleotide base, alkylated nucleotide bases, oxidizedbases (e.g., 8-hydroxyguanine), cross-linked nucleic acid strands,thymidine dimers, 6+4 photoproducts, nucleic acids with apurinic orapyrimidinic sites, etc).

Substrates, advantageously, have functional groups (e.g., amino groups,carboxylic acids, active esters, acid halides, hydroxyls, ketones,aldehydes, olefins, α,β-unsaturated carbonyls, a-halocarbonyls,hydrazides, imidazoles, thiols, disulfides, halides or other leavinggroups, photoactivatable groups, etc.) that allow for the convenientchemical linkage of the substrate to i) ECL labels, ii) solid phases,and/or iii) binding groups (e.g., biotin) that allow attachment tocomplementary binding groups (e.g., streptavidin) on a solid phase. Fora list of some useful linking chemistries and reagents, see the PierceChemical Company Catalog and Handbook for 1994-1995 (Pierce ChemicalCo., Rockford, Ill.) and Hermanson, Greg T. Bioconjugate TechniquesAcademic Press: New York, 1996, said publications hereby incorporated byreference. The labeling and/or immobilization of the substrate can beachieved by chemical treatment of the substrate molecule with functionalgroups present on the label and/or solid phase (e.g., amino groups,carboxylic acids, active esters, acid halides, hydroxyls, ketones,aldehydes, olefins, α,β-unsaturated carbonyls, α-halocarbonyls,hydrazides, imidazoles, thiols, disulfides, halides, photoactivatablegroups, etc.). By using standard coupling chemistries known in the art,it is possible to conveniently label/immobilize the natural substrate ofan enzyme (the cost, labor, and uncertainty of using unnatural orsynthetic substrates can be avoided). Control over the sites oflabeling/immobilization can be achieved by using coupling chemistriesspecific for a particular functionality on a substrate (e.g., anoligonucleotide that is 5′-modified with an amino group and 3′-modifiedwith a thiol group can be specifically labeled at the 5′ position withthe NHS ester of biotin, and specifically labeled at the 3′ positionwith a maleimide derivative of RuBpy). In an alternative embodiment, thesubstrate is chemically or enzymatically synthesized using labeled(and/or chemically modified) components so as to introduce labels(and/or points of attachment). For example, labeled or chemicallymodified amino acids can be introduced into defined positions of apolypeptide by solid phase synthesis. Similarly, labeled or chemicallymodified nucleotides (or phosphoramidites) can be introduced intodefined positions of a polynucleotide by solid phase synthesis. In oneembodiment, the solid phase support used in the synthesis of substratesby solid phase synthesis is also used as the solid phase of the ECLenzyme assay. In a different example, a polynucleotide substrate issynthesized through a polymerase reaction run in the presence of labeledand/or chemically modified nucleotides.

Cleaving Enzymes

The activity of cleaving enzyme is determined by measuring the effect ofthe enzyme on the concentrations or amounts of an ECL label in solutionor on a solid phase. FIGS. 5a-f illustrate different embodiments of theinvention. The illustrated embodiments are similar in that the enzyme501 cleaves at least one linkage in a substrate 502, the linkage linkingone or more ECL labels 503 with a solid phase (or alternatively a moietycapable of being immobilized on a solid phase) 504. The enzyme cancleave one linkage in the substrate (FIGS. 5a,c,e) or more than onelinkage (FIGS. 5b,d). The substrate can comprise one ECL label (FIGS.5a,b) or more than one ECL label (FIGS. 5d-f). The products can includemore than one solution-phase product comprising an ECL label (FIGS.5d-e) as well as solution-phase products not comprising an ECL label(FIG. 5b). An individual product can comprise more than one ECL label(FIG. 5c). The reactions can require additional substrates that are notshown and/or could form additional products that are not shown. FIG. 5fshows an assay for an enzyme with transferase activity; the cleavedportion of the substrate is transferred to a second substrate 505.

Joining Enzymes

The activity of a joining enzyme is also determined by measuring theeffect of the enzyme on the concentrations or amounts of an ECL label insolution or on a solid phase. FIGS. 6a-e illustrate differentembodiments of the invention. The illustrated embodiments are similar inthat the enzyme 601 forms at least one linkage joining substrates 602,the linkage linking one or more ECL labels 603 with a solid phase (oralternatively a moiety capable of being immobilized on a solid phase)604. The enzyme can form one linkage in the product (FIGS. 6a,c,e) ormore than one linkage (FIGS. 6b,d). The product can comprise one ECLlabel (FIGS. 6a,b) or more than one ECL label (FIGS. 6d-f). Thesubstrates can include more than one solution-phase substrate comprisingan ECL label (FIGS. 6d-e) as well as solution-phase substrates notcomprising an ECL label (FIG. 6b). An individual substrate can comprisemore than one ECL label (FIG. 6c). The reactions can require additionalsubstrates that are not shown and or form additional products that arenot shown.

Joining activity can be measured that joins a plurality of substrates toform a large aggregate. For example, thrombin can be measured by using asubstrate mixture containing fibrinogen labeled with an ECL label andfibrinogen linked to a solid phase (or alternatively to a capturemoiety). The enzyme activity converts the fibrinogen into a fibrin clotcomprising a plurality of fibrin monomers; the clot comprises both ECLlabels and links to one or more solid phases (or one or more capturemoieties). The thrombin activity is, therefore, directly related to thequantity of ECL labels on a solid phase or linked to a capture moiety.The fibrin clot dissociates in dilute acid, but the transglutaminase A₂crosslinks the fibrin clot to form an acid-stable clot. TransglutaminaseA₂ in a sample can, therefore, be measured by treating the sample with athrombin induced clot (prepared as in the abovementioned thrombin assay)as a substrate and measuring the quantity of ECL labels on the solidphase after treatment of the clot with dilute acid. Transglutaminase A₂also joins other proteins to fibrin during clot formation (e.g.,α₂-antiplasmin and Factor V). By analogy to the general scheme formeasuring joining enzymes, transgluminase can be measured, e.g., by itsactivity for joining fibrin labeled with an ECL-label and α₂-antiplasmininto a clot. See Blood: Principles and Practice of Hematology, R. Handinet al., Eds., J. B. Lippencott Co.: Philadelphia, 1995, herebyincorporated by reference, for more information on joining and cleavingenzymes present in blood).

Enzyme Assays

The invention can be used to assay an enzyme of interest. Generally,such an assay involves mixing a sample containing an unknown quantity ofthe enzyme with a predetermined quantity of one or more substrates anddetermining the amount or activity of the enzyme through a measurementof ECL. The invention can also be used to measure conditions or factorsthat can influence the activity of an enzyme, e.g., temperature, pH,enzyme inhibitors, denaturing compounds, enzyme activators, enzymedeactivators and the like.

Enzyme Inhibition Assays

The invention can also be used to assay an enzyme inhibitor and/or tomeasure the inhibitory ability of test compound. Generally, such anassay involves mixing a sample of unknown inhibitory ability (e.g., asample containing an unknown quantity of a known inhibitor or a samplecontaining a test compound) with a predetermined quantity of an enzymeand one or more substrates, and determining the ability of the sample toinhibit the enzyme through a measurement of ECL.

Assays for Enzyme Substrates

The invention can also be used to assay an enzyme substrate. Generally,such an assay involves mixing a sample containing an unknown quantity ofan enzyme substrate with a predetermined quantity of an enzyme (and, ifrequired, one or more additional substrates) and measuring the formationof product through an ECL measurement. The invention can also be used todetermine if a substance is accepted as a substrate by an enzyme.Generally, such an assay involves mixing a sample containing a possibleenzyme substrate with a predetermined quantity of an enzyme (and, ifrequired, one or more additional substrates) and measuring the formationof product through an ECL measurement.

Furthermore, the invention can be used to monitor a second reaction (inaddition to the enzyme reaction) that modulates the amount of an enzymesubstrate and/or the ability of a substrate to act as a substrate in theenzyme reaction. The ECL enzyme assay is used, e.g., to measure thepresence of substrates, products, or catalysts of the second reaction.In one such embodiment, the ability of a substance to act as a substratefor an enzyme is modulated as the result of a binding reaction (i.e.,said second reaction is a binding reaction). In the case of nucleicacids as substrates for nucleases, these binding reactions include: thebinding of a single stranded nucleic acid with a complementary nucleicacid sequence; the binding of a double stranded nucleic acid with atriple helix forming molecule such as a nucleic acid, a peptide nucleicacid (PNA), a minor or major groove binding peptide (e.g., distamycinand polyamides containing hydroxypyrrole, imidazole and/or pyrrole suchas those described in White et al. Nature, 1998, 391, 468, herebyincorporated by reference), and/or a nucleic acid binding protein. Inone example, the ability of a ribonuclease specific for single strandedRNA to cleave a first RNA sequence is modulated by the presence of acomplementary sequence that can participate in a hybridization reactionwith the first sequence; this effect provides the basis for themeasurement of the complementary sequence in a sample. In a secondexample, the binding of a nucleic acid binding protein to a nucleic acidcan block the ability of a nuclease to cleave the nucleic acid; thiseffect can be used to determine the quantity, binding ability, orspecificity of a nucleic acid binding protein in a sample. The effectcan also be used to measure the ability of a sample (or components of asample) to inhibit the interaction between a nucleic acid and a nucleicacid binding protein.

Enzymes (e.g., proteases specific for phosphorylated proteins orpeptides, protesases specific for ubiquitinated proteins or peptides,DNA repair enzymes, etc.), that take modified nucleic acids or proteinsas substrates (e.g., phosphorylated amino acids, ubiquinated aminoacids, methylated nucleotide base, alkylated nucleotide bases, oxidizedbases such as 8-hydroxyguanine, cross-linked nucleic acid strands,thymidine dimers, 6+4 photoproducts, nucleic acids with apurinic orapyrimidinic sites, etc.) can be used to measure these substrates and,therefore, to measure enzymes, drugs, reagents, toxins, and/orconditions that cause or repair these modifications (e.g., the causationor repair of said modifications can be the above mentioned secondreaction).

Kits for Carrying out the Methods of the Invention

The invention also includes reagents and kits for carrying out themethods of the invention. A kit for carrying out the methods of theinvention can comprise, in one or more containers, at least two of thefollowing components: enzyme, substrate, solid phase, buffersappropriate for carrying out the enzymatic reaction (e.g., mixtures ofpH buffering substances, detergents, salts, metal ions, cofactors,proteins, sugars, excipients, and the like), solutions appropriate forcarrying out an ECL measurement, solutions appropriate for cleaningand/or conditioning an ECL measuring device, calibration solutionscontaining known concentrations of an enzyme, calibration solutionscontaining a known concentration of an enzyme inhibitor, and calibrationsolutions for calibrating the response of an ECL measuring instrument.These components can be supplied in dry and/or liquid form. Kits formeasuring the interaction between nucleic acids and a nucleic acidbinding protein can additionally include one or more of the followingcomponents: nucleic acid substrate, nucleic acid binding protein,calibration solutions containing known amounts of a DNA binding protein,calibration solutions containing a known amount of a nucleic acidsubstrate, or calibration solutions containing a known amount of aninhibitor of a protein-nucleic acid interaction.

Assay Formats

A. Method for Assayinz a Sample for the Presence of a Nucleic AcidBinding Protein

In one embodiment of the present invention, the interaction of a nucleicacid binding protein with a nucleic acid is measured using a nuclease orchemical protection approach. This approach makes use of the ability ofa nucleic acid binding protein, when bound to a nucleic acid, to protect(fully or partially) the phosphodiester backbone of the nucleic acidfrom cleavage by a nucleic acid cleaving enzyme or reagent. The bindingof the protein to the nucleic acid can be monitored by measuring theextent of the cleavage reaction (e.g., by measuring the decrease in theamount of substrate and/or the increase in the amount of product). Theprotection of a nucleic acid by a nucleic acid binding protein can beused to measure the amount of a nucleic acid binding protein in asample, to measure the affinity of a binding protein for a nucleic acidsequence, to screen for peptides or proteins capable of binding aspecific sequence, to screen for a nucleic acid sequence that is boundby a specific protein, and/or to screen for inhibitor substances thatinhibit the interaction. The method is easily adaptable to use in highthroughput screening. Libraries of compounds (e.g., substrates,proteins, peptides, inhibitors) include libraries of more than 100compounds, or advantageously, libraries of more than 10,000 compounds,or most advantageously, libraries with more than 1,000,000 compounds.

The proteins or peptides that can be measured by this method include,but are not limited to, proteins that bind nucleic acids to regulatenucleic acid translation, transcription, reproduction, editing,localization, degradation, repair, etc. They also include triplehelix-forming nucleic acid sequences, nucleic acid binding toxins,antibiotics, and regulatory proteins from bacteria, viruses, and othersources. The proteins or peptides can be from natural sources orman-made. The proteins can bind to specific nucleic acid sequences oralternatively can have limited or no sequence specificity.

Advantageously, the cleaving enzyme or reagent shows a greater than 20fold preference for the unprotected nucleic acid vs. the protectednucleic acid. Enzymatic cleavage reagents (e.g. nucleases) can beselected by screening for enzymes according to their selectivity for theunprotected nucleic acid substrate of an assay. Non-enzymatic cleavagereagents include transition metal complexes capable of oxidizing nucleicacid (advantageously linked to a nucleic acid binding moiety orintercalater), photoactivated cleaving reagents, acids, bases, and thereagents used for cleaving DNA in Maxam-Gilbert sequencing (e.g.,dimethylsulfate/heat/alkali, dimethylsulfate/acid/alkali,hydrazine/piperidine). In an alternate embodiment, the nucleic acidbinding protein protects the nucleic acid from a chemical, enzymaticand/or photochemical modification for example, methylation, alkylation(e.g., by a cancer therapeutic agent), oxidation (e.g., to form8-hydroxyguanine), base excision, strand cross linking, formation ofthymidine dimers, formation of 6+4 photoproducts, etc.) that make thenucleic acid more susceptible to a second enzyme or reagent that, e.g.,cleaves nucleic acids (e.g., a methylated nucleic acid specific nucleaseor an AP endonuclease).

The predetermined (except in the case of an enzyme activity with randomspecificity) nucleic acid substrates can be single or double stranded orcomprise regions of both. Advantageously, the nucleic acid substrate is4-1000 kilobases in length, more advantageously 4-100 kilobases inlength, and most advantageously 4-30 kilobases in length. The nucleicacid substrate comprises a protein binding site, for example a sequencespecific for a protein of interest; the substrate also comprises a sitecapable of being cleaved by a nucleic acid cleaving enzyme or reagent.Advantagously, the binding protein, nucleic acid substrate, and/or thecleaving enzyme/reagent are chosen so that the cleavage site lies withinthe protein binding site and the protective effect is maximized. In somecases there can be additional cleavage sites (e.g., sites fallingoutside of the protein binding site)) that are not sufficientlyprotected by the binding protein. These additional cleavage sites areadvantageously protected from cleavage by the use of nucleic acidanalogs that are resistant to cleavage, e.g., nucleotides that arelinked by amide bonds (e.g., peptide nucleic acids), phosphorothioatebonds, or methyl phosphonate bonds. In an advantageous embodiment, theregion of the nucleic acid within the protein binding site comprisesnucleotides linked by phosphodiester bonds (advantageously such a regioncomprises from 2-100, more advantageously from 2-50, and mostadvantageously 4-14 nucleotides linked by phosphodiester bonds); theregion outside the protein binding site comprises nucleotides linked byphosphorothioate bonds. Advantageously, a nucleic acid containingunnatural nucleotides comprises from 1 to 999 unnatural nucleotides.

The measurements of the decrease in the amount of substrate or theincrease in the amount of product can be measured by any technique thatcan be used to measure a nucleic acid of e.g., a particular size,sequence, composition, and/or charge. For example substrates or productscan be measured by chemical analysis (e.g., mass spectrometry,chromatography, electrophoresis, NMR, nucleic acid sequencing, etc.) orby hybridization (e.g., Northern blots, southern blots, solid phasebinding assays, fluorescence energy transfer methods such as the use ofmolecular beacons, etc.—see, e.g., Nonradioactive Labeling and Detectionof Molecules, Kessler, C., ed., Springer-Verlag: Berlin, 1992 andKeller, G. H.; Manak, M. M. DNA Probes, 2nd Ed., MacMillan PublishersLtd.: London, 1993, each of these books, hereby, incorporated byreference).

Nucleic Acid Substrates that Comprise a Detectable Label and are Linked(or Capable of Being Linked) to a Solid Phase.

In one embodiment of the invention's assays for nucleic acid-proteininteractions, the nucleic acid substrate comprises one or moredetectable labels and is also linked to a solid phase (or,alternatively, comprises one or more moieties capable of being capturedat a solid phase, e.g., biotin, a specific nucleic acid sequence, ahapten, a ligand, etc.). The nucleic acid substrate is constructed insuch a way that one or more of the labels are separated from the solidphase (or, alternatively, the capture moieties) by a sequence ofnucleotides that comprise both a protein binding sequence and a sitecapable of being cleaved by a nuclease. The regions of the nucleic acidoutside the protein binding site can advantageously comprise nucleotideslinked by cleavage-resistant linkages (e.g., phosphorothioate linkages)to prevent cleavage of the nucleic acid in regions that can't beprotected by the nucleic acid binding protein. In a protein bindingassay, the nucleic acid substrate is mixed with the protein sample,advantageously in a buffer that promotes the association of the proteinwith the nucleic acid. The sample is subsequently mixed with a nucleicacid-cleaving enzyme, advantageously for a defined length of time and ata defined temperature. The amount of the detectable label on the solidphase or free in solution is then measured (if necessary, aftercapturing the capture moieties by contacting the mixture with a solidphase) to determine the extent of substrate cleavage and therefore theamount of protein-nucleic acid complex that formed. The binding of theprotein to the nucleic acid results in an increase in the label on thesolid phase and a decrease in the label in solution. Detectable labelsthat can be used include, but are not limited to, enzymes,radioisotopes, fluorescent labels, chemiluminescent labels, ECL labels,bioluminescent labels, electrochemically detectable labels, magneticlabels, optically detectable particles such as colloidal gold, etc. Inone embodiment of the invention, an array of labeled nucleic acids isimmobilized on a solid phase (advantageously an electrode), e.g., todetermine the consensus sequence of one or more nucleic acid bindingproteins in a sample. In this example, detectable labels on arrayelements comprising a consensus sequence are protected from treatmentwith a nuclease and remain largely on the solid phase, while detectablelabels on array elements without a consensus sequence are cleaved off bytreatment with a nuclease.

Nucleic Acid Substrates that Comprise an ECL Label and are Linked (orCapable of Being Linked) to a Solid Phase.

In a advantageous embodiment of the invention's assays for nucleicacid-protein interactions (illustrated in FIG. 7), the nucleic acidsubstrate comprises one or more ECL labels and is also linked to a solidphase (or, alternatively, comprises one or more moieties capable ofbeing captured at a solid phase, e.g., biotin, a specific nucleic acidsequence, a hapten, a ligand, etc.). Advantageous ECL labels includeluminol and bipyridyl- or phenanthrolyl-containing complexes of Ru, Osand Re. An especially advantageous label is RuBpy. The nucleic acidsubstrate is constructed in such a way that one or more of the labelsare separated from the solid phase (or, alternatively, the capturemoieties) by a sequence of nucleotides that comprise both a proteinbinding sequence and a site capable of being cleaved by a nuclease. Theregions of the nucleic acid outside the protein binding site canadvantageously comprise nucleotides linked by cleavage-resistantlinkages such as phosphorothioate linkages to prevent cleavage of thenucleic acid in regions that can't be protected by the nucleic acidbinding protein. In a protein binding assay, the nucleic acid substrateis mixed with the protein sample, advantageously in a buffer thatpromotes the association of the protein with the nucleic acid. Thesample is subsequently mixed with a nucleic acid-cleaving enzyme,advantageously for a defined length of time and at a definedtemperature. The amount of the detectable ECL label on the solid phaseor free in solution is then measured (if necessary, after capturing thecapture moieties by contacting the mixture with a solid phase) todetermine the extent of substrate cleavage and therefore the amount ofprotein-nucleic acid complex that formed. The binding of the protein tothe nucleic acid results in an increase in the label on the solid phaseand a decrease in the label in solution. In one advantageous embodiment,the solid phase is a magnetic bead and the ECL label on the solid phaseis measured after using a magnetic field to capture the beads on anelectrode for inducing ECL, e.g., through the use of an ORIGEN analyzer.In a different advantageous embodiment, the solid phase is an electrode(e.g., a composite comprising carbon nanotubes in polymeric matrix) andthe ECL label on the solid phase is measured by applying a potential atthe electrode so as to induce the ECL labels to electrochemiluminesce.These measurements of ECL labels are, highly quantitative, sensitive,and precise. Advantageously the assay has a detection limit (formeasuring a nucleic acid binding protein, its nucleic acid partner, oran inhibitor of the interaction) of less than 1 nmol, moreadvantageously, the detection limit is less than 1 pmol, even moreadvantageously, the detection limit is less than 1 fmol, even moreadvantageously, the detection limit is less than 1 amol.

Nucleic Acid Substrates for Measuring DNA-Protein Interactions by theFluorescence Resonance Energy Transfer (FRET) Technique.

In another advantageous embodiment of the invention's assays for nucleicacid-protein interactions, the nucleic acid substrate is linked to oneor more fluorescence energy donors and one or more fluorescence energyacceptors (one with skill in the art of FRET assays can selectappropriate donors and acceptors and appropriate substrate structuresfor a particular assay so as to produce efficient energy transfer fromdonor to acceptor). In one embodiment of a FRET assay for measuringprotein-nucleic acid interactions, the nucleic acid substrate isconstructed in such a way that at least one of the donors is separatedfrom at least one of the acceptors by a sequence of nucleotides thatcomprise both a protein binding sequence and a site capable of beingcleaved by a nuclease. The regions of the nucleic acid outside theprotein binding site can advantageously comprise nucleotides linked bycleavage-resistant linkages such as phosphorothioate linkages to preventcleavage of the nucleic acid in regions that can't be protected by thenucleic acid binding protein. In a protein binding assay, the nucleicacid substrate is mixed with the protein sample , advantageously in abuffer that promotes the association of the protein with the nucleicacid. The sample is subsequently mixed with a nucleic acid-cleavingenzyme, advantageously for a defined length of time and at a definedtemperature. The cleavage of the substrate leads to a decrease in theamount of fluorescence energy transfer due to an increase in thedistance between the donor and acceptor moieties. This decrease inenergy transfer can be measured by measuring the increase in thefluorescence intensity from the donor and/or by measuring the decreasein intensity of the acceptor (resulting from excitation of the donor).The more nucleic acid binding protein that is present, the less cleavageis observed and, therefore, the lower the fluorescence signal due to thedonor and the higher the fluorescence signal due to the acceptor.

B. Method for Assaying a Sample for the Presence of an Enzyme Activitythat Joins Nucleic Acids

In another embodiment of the present invention, an enzyme of interestthat forms nucleic acid linkages between nucleic acids and/ornucleotides is measured in a sample by combining the sample with atleast one, advantageously two, predetermined (except in the case of anenzyme activity with random specificity) nucleic acid (and/ornucleotide) substrates, wherein at least one of said substratescomprises one or more ECL labels (advantageously luminol or bipyridyl-or phenanthroline-containing complexes of Ru or Os, most advantageouslyRuBpy) and at least one other of said substrate is linked to a solidphase (advantageously, a magnetic bead or an electrode). The nucleicacid substrates can be single or double stranded or comprise regions ofboth. . Advantageously, the nucleic acid substrate is 4-1000 kilobasesin length, more advantageously 4-100 kilobases in length, and mostadvantageously 4-30 kilobases in length. The substrates are designed orprepared so that the enzymatic reaction links at least one ECL label ona substrate to a solid phase. The extent of enzymatic joining isdetermined by a measurement of the ECL labels that couple to the solidphase. Increased enzymatic activity leads to an increase in the couplingof the ECL labels to the solid phase and, therefore, an increase in ECLsignal. Alternatively, the extent of enzymatic cleavage can bedetermined by a measurement of the ECL labels that remain free insolution (e.g., the ECL from ECL labels in solution can bepreferentially measured at an electrode in the presence of ECL labelspresent on particulate solid phases in suspension). In this alternativeembodiment, increased enzymatic activity results in a decrease in ECLsignal.

These measurements of ECL labels are highly quantitative, sensitive, andprecise. Advantageously the assay has a detection limit (for measuring anucleic acid cleaving enzyme, its substrates, or an inhibitor of theenzyme) of less than 1 nmol, more advantageously, the detection limit isless than 1 pmol, even more advantageously, the detection limit is lessthan 1 fmol, even more advantageously, the detection limit is less than1 amol.

Advantageously, the enzyme sample is contacted with the nucleic acidand/or nucleotide substrates for a defined period of time under definedconditions (e.g., temperature, pH, etc.) prior to the ECL measurement.In alternate embodiments, the nucleic acid and/or nucleotide substratesdo not include substrates that are linked to a solid phase but includesubstrates that comprise moieties that can be captured on a solid phase(e.g., biotin, specific nucleic acid sequences, ligands or haptens). Thesubstrate can be captured on a solid phase by contacting the substratewith a solid phase comprising groups capable of binding to saidmoieties; this contacting can be accomplished prior to, during, and/orafter the sample is contacted with the enzyme sample. In a differentembodiment, the substrate is non-specifically captured on a solid phase.

Classes of nucleic acid joining enzymes that can be measured includepolymerase, enzymes that covalently join nucleic acid moleculesincluding proteins involved with DNA recombination (e.g. integrases &recombinases), as well as DNA and RNA ligases.

An example of a format of the type mentioned herein is that of a strandtransfer assay for the enzyme integrase. In this format, aviral-specific donor DNA sequence that comprises a biotin is attached tomagnetic beads via a biotin-streptavidin interaction. This configurationallows one to pre-bind. the donor with integrase and wash the unboundenzyme prior to the addition of target DNA linked to RuBpy. Theintegrase first randomly nicks the RuBpy-labeled target molecules. DNAstrand transfer catalyzed by integrase leads to the formation of acovalent bond between the 3′ end of the biotinylated donor molecule andthe 5′ end of the nicked RuBpy-labeled target molecule leading to anincrease in the ECL signal from RuBpy on the magnetic beads, as measuredusing an ORIGEN analyzer.

C. Method for Assaying a Sample for an Enzyme Activity that CleavesNucleic Acids that Results in a Decrease in ECL Signal

In an embodiment of the present invention, an enzyme of interest thatcleaves nucleic acids (e.g., a nuclease, Dnase, Rnase, or restrictionendonuclease) is measured in a sample by combining the sample with apredetermined (except in the case of an enzyme activity with randomspecificity) nucleic acid substrate capable of being cleaved by theenzyme of interest, wherein said substrate comprises one or more ECLlabels (advantageously bipyridyl- or phenanthroline-containing complexesof Ru or Os, most advantageously RuBpy) and said substrate is linked toa solid phase (advantageously a magnetic bead or an electrode). Theenzyme can be specific for a specific nucleic acid sequence orstructure, or can have limited or no specificity. The nucleic acidsubstrate can be single stranded or double stranded or have regions ofboth. . Advantageously, the nucleic acid substrate is 4-1000 kilobasesin length, more advantageously 4-100 kilobases in length, and mostadvantageously 4-30 kilobases in length. The substrate is designed orprepared so that the enzymatic reaction decouples at least one ECL labelon a substrate molecule (advantageously, all the ECL labels on thesubstrate) from the solid phase. The extent of enzymatic cleavage isdetermined by a measurement of the ECL labels remaining on the solidphase. Increased enzymatic activity leads to an increase in thedecoupling of the ECL labels from the solid phase and, therefore, adecrease in ECL signal. Alternatively, the extent of enzymatic cleavagecan be determined by a measurement of the ECL labels released from thesolid phase into solution (e.g., the ECL from ECL labels in solution canbe preferentially measured at an electrode in the presence of ECL labelspresent on particulate solid phases in suspension). In this alternativeembodiment, increased enzymatic activity results in an increase in ECLsignal.

These measurements of ECL labels are highly quantitative, sensitive, andprecise. Advantageously the assay has a detection limit (for measuring anucleic acid cleaving enzyme, its nucleic acid substrate, or aninhibitor of the enzyme) of less than 1 nmol, more advantageously, thedetection limit is less than 1 pmol, even more advantageously, thedetection limit is less than 1 fmol, even more advantageously, thedetection limit is less than 1 amol.

Advantageously, the sample is contacted with the nucleic acid substratefor a defined period of time under defined conditions (e.g.,temperature, pH, etc.) prior to the ECL measurement. In alternateembodiments, the protein or peptide substrate is not linked to a solidphase but comprises moieties that can be captured on a solid phase(e.g., biotin, specific nucleic acid sequences, or haptens); one ofthese embodiments in illustrated in FIG. 8. The substrate can becaptured on a solid phase by contacting the substrate with a solid phasecomprising groups capable of binding to said moieties; this contactingcan be accomplished prior to, during, and/or after the sample iscontacted with the enzyme sample. In a different embodiment, thesubstrate is non-specifically captured on a solid phase.

A protocol for an ECL-based assay for measuring the enzymatic cleavageof viral specific sequences by viral integrase is described in detailbelow. Integrase is a retroviral enzyme that possesses several distinctcatalytic activities including those that promote processing and strandtransfer functions. The processing activity functions to cleave the DNAcopy of the retroviral genome in a sequence-specific fashion between theGA and CT bases of a GACT sequence. This activity is necessary for theintegration of the viral DNA into the host genome. The assay measuresthe cleavage of a double stranded nucleic acid substrate (advantageouslyhaving a length of between 18-30 bases) comprising a GACT sequence onone of the 3′ ends, a biotin on one end, and a RuBpy label on the otherend.

The substrate (advantageously, at a concentration of between 0.1 and 200uM) in a buffered solution (advantageously, at a pH of between 6.5-8.0and containing a cation such as manganese or magnesium at aconcentration of between 10-100 mM) is combined with the integrasesample and incubated (advantageously, at a temperature between 24-37° C.for between 5-1000 min.). Streptavidin-coated magnetic particles(advantagously, between 5-50 ug or Streptavidin DynaBeads) are thenadded and the suspension mixed for 10 min. The ECL from RuBpy remainingon the magnetic beads or released into solution is measured on an ORIGENanalyzer (IGEN International) running, respectively, in magnetic captureor solution phase modes. The integrase activity is directly related tothe ECL from RuBpy released into solution and correlates with a drop inECL from RuBpy on the magnetic beads.

D. Method for Assaying a Sample for the Presence of an Enzyme Activitythat Cleaves Peptides or Proteins

In another embodiment of the present invention, an enzyme of interestthat cleaves peptides or proteins (e.g., a protease or a peptidase) ismeasured in a sample by combining the sample with a predetermined(except in the case of an enzyme activity with random specificity)protein or peptide substrate capable of being cleaved by the enzyme ofinterest, wherein said substrate comprises one or more ECL labels(advantageously, bipyridyl- or phenanthroline-containing complexes of Ruor Os, most advantageously RuBpy) and said substrate is linked to asolid phase (advantageously a magnetic bead or an electrode). The enzymecan be specific for a specific protein or peptide sequence or structure,or can have limited or no specificity. The substrate is designed orprepared so that the enzymatic reaction decouples at least one ECL labelon a substrate molecule (advantageously all the ECL labels on thesubstrate) from the solid phase. Advantageously, the substrate is 4-1000amino acids in length, more advantageously 4-100 amino acids in length,and most advantageously 4-40 amino acids in length. The extent ofenzymatic cleavage is determined by a measurement of the ECL labelsremaining on the solid phase. Increased enzymatic activity leads to anincrease in the decoupling of the ECL labels from the solid phase and,therefore, a decrease in ECL signal. Alternatively, the extent ofenzymatic cleavage can be determined by a measurement of the ECL labelsreleased from the solid phase into solution (e.g., the ECL from ECLlabels in solution can be preferentially measured at an electrode in thepresence of ECL labels present on particulate solid phases kept insuspension). In this alternative embodiment, increased enzymaticactivity results in an increase in ECL signal.

These measurements of ECL labels are highly quantitative, sensitive, andprecise. Advantageously the assay has a detection limit (for measuring aprotein cleaving enzyme, its substrate, or an inhibitor of the enzyme)of less than 1 nmol, more advantageously, the detection limit is lessthan 1 pmol, even more advantageously, the detection limit is less than1 fmol, even more advantageously, the detection limit is less than 1amol.

Advantageously, the sample is contacted with the protein or peptidesubstrate for a defined period of time under defined conditions (e.g.,temperature, pH, etc.) prior to the ECL measurement. In alternateembodiments, the protein or peptide substrate is not linked to a solidphase but comprises moieties that can be captured on a solid phase(e.g., biotin, specific nucleic acid sequences, or haptens). Thesubstrate can be captured on a solid phase by contacting the substratewith a solid phase comprising groups capable of binding to saidmoieties; this contacting can be accomplished prior to, during, and/orafter the sample is contacted with the enzyme sample. In a differentembodiment, the substrate is non-specifically captured on a solid phase.

E. Method for Assaying for the Presence of a Specific Nucleic AcidSequence

In another embodiment of the present invention, a nucleic acid sequenceof interest in a sample is measured by combining the sample with apredetermined (with respect to at least 10 nucleotides) nucleic acidprobe (advantageously 8 to 10,000 nucleotides, more advantageously 15 to1000 nucleotides) comprising a sequence complementary (or partiallycomplementary) to the sequence of interest, said probe comprising an ECLlabel (advantageously a bipyridyl- or phenanthroline-containing complexof Ru or Os, most advantageously RuBpy) and a moiety capable of beingcaptured on a solid phase (advantageously, biotin, a specific nucleicacid sequence, or a hapten). This combining is advantageously carriedout in a buffer solution and a temperature (advantageously 0-100° C.,most advantageously, 4-70° C.) that promotes the specific hybridizationof the sequence of interest with its complementary sequence. The sampleis then incubated with a nucleic acid-cleaving enzyme specific forsingle-stranded nucleic acid molecules (e.g., RNase A, mung beannuclease, or nuclease S₁). The resulting mixture is contacted with asolid phase capable of capturing said moiety and the captured ECL labelsare measured by ECL (e.g., on an ORIGEN analyzer, IGEN International.The ECL signal increases with the amount of labeled nucleic acidhybridized to the sequence of interest and thus serves as a directmeasure of the quantity of specific sequence found in the nucleic acidsample preparation. In an alternate embodiment, the ECL labeled probe isdirectly linked to a solid phase and the solid phase is present duringthe binding and cleavage reactions. In another alternate embodiment, anuclease specific for double stranded nucleic acids is used (e.g.,nuclease BAL 31 or exonuclease III); in this embodiment, specifichybridization leads to a decrease in ECL signal from ECL labels on thesolid phase.

These measurements of ECL labels are highly quantitative, sensitive, andprecise. Advantageously the assay has a detection limit (for measuring anucleic acid sequence) of less than 1 nmol, more advantageously, thedetection limit is less than 1 pmol, even more advantageously, thedetection limit is less than 1 fmol, even more advantageously, thedetection limit is less than 1 amol.

The following Examples are illustrative, but not limiting of thecompositions and methods of the present invention. Other suitablemodifications and adaptations of a variety of conditions and parametersnormally encountered which are obvious to those skilled in the art arewithin the spirit and scope of this invention.

EXAMPLES Example 1 A DNase Protection Assay for the Assessment ofDNA-protein Interactions

This example illustrates the use of the invention to measure protein-DNAinteractions, specifically, the binding of the transcription factor NFkβwith its consensus DNA sequence. A schematic depicting the assay formatcan be found in the detailed description of the invention. DNAse 1 andNFkβ were purchased from Promega Corp. A DNA substrate containing aconsensus DNA sequence for NFk was prepared by Midland Certified ReagentCo. by solid phase synthesis. The sequence of the substrate is shownbelow. The nucleotides shown in brackets were linked by phosphorothioatelinkages; the other linkages were standard phosphodiester bonds. One ofthe strands of the double stranded DNA substrate was labeled at the5′-end with RuBpy and at the 3′-end with biotin using standard labelingtechniques.

5′-RuBpy-[AGTTGAGG]GGACTTT[CCCAGGC]-Biotin-3′ (SEQ ID NO 1)TCAACTCCCCTGAAAGGGTCCG-3′ (SEQ ID NO 2)

The labeled DNA substrate (50 fmol), poly dI-dC (1 ug, to reducenon-specific protein-DNA interactions) and varying amounts ofrecombinant NFkβ (p50 subunit) were combined in a 20 uL volume andincubated for 30 min. at room temperature. Dnase 1 (2 Units in a volumeof 2 uL, Promega Corp.) was then added and the incubation was continuedfor an additional 30 min. at room temperature. The reaction wasterminated and the biotin-labeled DNA sequences captured by in theaddition of 10 μg of streptavidin Dynabeads (IGEN, International) in 0.3ml PBS-1 containing 0.2 M EDTA, followed by incubation for 15 minutes.The reaction mixture was introduced into an ORIGEN Analyzer (IGENInternational) running in Magnetic Capture Mode and the ECL signal fromRuBpy on the magnetic beads was determined in the presence of a solutioncontaining tripropylamine (ORIGEN Assay Buffer, IGEN International). Acontrol was also run in the absence of Dnase to determine the ECL signalobtained from the uncleaved substrate. Table 1 shows that the additionof Dnase to unprotected DNA gave a >20 fold reduction in signal. Theaddition of NFkβ gave a dose dependent increase in signal relative tothat obtained from unprotected DNA, the magnitude of which approached,for high concentrations of NFkβ, the signal obtained in the absence ofDNAse. The experiment was repeated with an DNA substrate with the samenucleotide sequence but containing only phosphodiester linkages andsimilar results were obtained.

TABLE I Dnase NFkB (p50 subunit) ECL Signal 0 0 2591957 2 U 0 96862 2 U7 ng 1729305 2 U 3.5 ng 1383572 2 U 1.75 ng 709996 2 U 0.87 ng 425194

The specificity of the assay was determined by replacing the specificbinding protein NFkβ with other DNA binding proteins specific forsequences not present on the DNA substrate (the phosphorothioatecontaining substrate was used). Table II shows only the specific bindingprotein, NFkβ, was able to confer nuclease protection to the DNAsubstrate containing the NFkβ consensus sequence. The DNA bindingproteins AP-1, AP-2, and SP-1 showed no ability to protect againstnuclease attack.

TABLE II Transcription Factor DNase Added ECL Signal None No 2334309None Yes 54041 NFkB Yes 1209953 AP-1 Yes 32303 AP-2 Yes 32289 SP-1 Yes38880

The DNAse protection assay was able to specifically detect the bindingof the NFkβ consensus sequence to NFkβ present in nuclear extracts. Wereplaced the recombinant NFkβ used in the previous experiments with theNFkβ activity present in 1 uL of the nuclear extract from HeLa cells(Promega Corp.). Table III gives the specific ECL signal obtained fromthe assay (given as the difference between the ECL signal for the assayand the ECL signal measured for unprotected DNA) and shows that thenuclear extract was able to protect the DNA substrate from cleavage. Theprotection was due to a specific interaction between NFkβ and itsconsensus sequence. Table III also shows that a specific competitor ofthe interaction (a 100 fold excess of unlabeled DNA containing the NFkβconsensus sequence) reversed the protective effect of the cellularextract. In contrast, non specific sequences (100 fold excesses ofunlabeled DNA containing the consensus sequences for AP-1 or SP-1) hadno effect. The use of a DNA substrate having only phosphodiester bonds(i.e., no phophorothioate) gave similar specificity although thespecific ECL signals were lower.

TABLE III Determination of NFkβ Binding Activity in HeLa Cell ExtractCompetitor Sequence Used Specific ECL Signal None 744155 SP-1 665826AP-1 829931 NFkB 0

A similar experiment was conducted using a dual labeled SP-1 consensuswhose sequence is given below.

5′-Ruthenium-GATCGAACTGACCGCCCGCGGCCCGT-Biotin-3′ (SEQ ID NO 3)CTAGCTTGACTGGCGGGCGCCGGGCA (SEQ ID NO 4)

Table IV not only show the ability to measure SP-1 binding activity in acomplex protein preparation, but also demonstrates that only thespecific competitor sequence was able to successfully compete in thebinding reaction.

TABLE IV Determination of SP-1 Binding Activity in HeLa Cell ExtractCompetitor Sequence Used ECL Signal None 1385846 SP-1 0 AP-1 1172548NFkB 1323636

Example 2 An ECL-based Assay for the Measurement of Protease Activity

This assay system consists of a ruthenylated (RuBpy-labeled) substrateimmobilized on paramagnetic beads and the enzyme of interest. The RuBpylabel is released by the action of the enzyme. The ECL of the free labelis measured using the ORIGEN Analyzer (IGEN International).

Dynabead® M280 Sheep anti-mouse IgG coated beads (IGEN International,Inc.) were RuBpy-labeled at a 200:1 challenge ratio of RuBpy to IgG tointroduce RuBpy groups on the immobilized IgG molecules. The labelingwas carried out using a derivative of RuBpy linked to an NHS ester(TAG-NHS, IGEN International) according to established procedures. Thebeads were then washed three times, thirty minutes each, at 4° C. withequal volumes of PBS, pH 7.8, and once overnight at 4° C. Replicate testsamples were prepared with 100 μl of solutions containing known amountsof Proteinase K (Sigma) in phosphate buffered saline, pH 7.8 and 25 μlof 1.2 mg/ml TAG labeled Sheep anti-mouse beads. The samples were shakenfor 30 minutes at 37° C. The reaction was quenched by the addition of 1mL of a solution containing tripropylamine (ORIGEN Assay Buffer, IGENInternational, Inc.) and the protease activity was quantitated on theORIGEN 1.5 Analyzer with the solution phase default settings (i.e., thebeads are kept in suspension so as to preferentially measure ECL at theelectrode from RuBpy groups in solution) except for high vortex speed.FIG. 9 shows the ECL signal as a fuiction of the concentration ofProteinase K; the figure shows that the ECL signal is directly relatedto the concentration of enzyme.

Example III An ECL-Based Assay for the Measurement of Factor Xa Activity

Factor Xa is a serine protease that cleaves the site adjacent to thearginine in the amino acid sequence IEGRX. The assay uses a peptidesubstrate that is labeled at the N-terminus with RuBpy and at a lysineat the carboxy terminus with biotin (RuBpy-IEGRGUEUEK-Biotin).Streptavidin-coated Dynabeads (IGEN International) were used to capturethe labeled-peptide. The captured peptide is incubated with a samplecontaining the protease. Increasing amounts of protease in the sampleled to increased rates of cleavage and (for a given amount of time) lessRuBpy on the beads and more RuBpy in solution. The reaction productswere analyzed on an ECL measurement instrument (ORIGEN Analyzer, IGENInternational). The measurements were carried out in Solution Mode(i.e., the samples were analyzed under conditions that did not lead tosignificant settling of the bead suspension on the electrode; underthese conditions the ECL signal was primarily due ECL labels releasedinto solution and increased with increasing protease activity). Themeasurement can, alternatively, be carried out in Bead Capture Mode(i.e., a magnetic field is used to collect the magnetic beads arecollected on the electrode surface; under these conditions, the ECLsignal is primarily due to ECL labels on the solid phase and decreaseswith increased protease activity).

The protocol used was as follows: Factor Xa protease (1.28 Units in 5uL) was combined with 20 uL of streptavidin Dynabeads (precoated withlabeled substrate) and 175 uL of reaction buffer (50 mM tris, pH 8.0,200 mM NaCl, 6 mM CaCl₂) and incubated at 37° C. for varying amounts oftime. At the end of the predetermined incubation time, a 5 uL aliquot ofthe reaction mixture was combined with 345 uL of a tripropylaminecontaining buffer (ORIGEN Assay Buffer, IGEN International) and themixture analyzed on an ORIGEN analyzer in Solution Mode. As a negativecontrol, the experiments were repeated for the same times withoutprotease. For an incubation time of 0 min. (i.e., the reaction was notallowed to occur), the measured ECL signal was approximately the same asthe negative control. After 45 min. of incubation, the ratio of the ECLsignal to that of the negative control was 4:1.

It will be readily apparent to those skilled in the art that numerousmodifications and additions may be made to both the present invention,the disclosed device, and the related system without departing from theinvention disclosed.

4 1 22 DNA Artificial Sequence misc_difference (1)..(8) Nucleotides arelinked by phophorothioate linkages 1 agttgagggg actttcccag gc 22 2 22DNA Artificial Sequence Description of Artificial Sequence Artificialsequence containing consensus sequence for human NFkB 2 gcctgggaaagtcccctcaa ct 22 3 26 DNA Artificial Sequence Description of ArtificialSequenceArtificial sequence containing consensus sequence for human SP-13 gatcgaactg accgcccgcg gcccgt 26 4 26 DNA Artificial SequenceDescription of Artificial Sequence Artificial sequence containingconsensus sequence for human SP-1 4 acgggccgcg ggcggtcagt tcgatc 26

What is claimed is:
 1. A method for assaying a sample for the presenceof a nucleic acid binding protein, which comprises: a) mixing at leastone predetermined single- or double-stranded nucleic acid containing atleast one label and containing a protein binding nucleotide sequencewith a sample which may contain a nucleic acid binding protein, b)incubating the mixture of step a) under conditions which allow thebinding of said nucleic acid binding protein to said at least onepredetermined single- or double-stranded nucleic acid to form a complex,c) adding a nucleic acid-cleaving enzyme or reagent to the mixture ofstep b), d) incubating the mixture of step c) under conditions whichallow the cleavage of said at least one predetermined single- ordouble-stranded nucleic acid which has not formed a complex, and e)measuring the amount of said complex by means that do not include gelelectrophoretic separation to measure said nucleic acid binding protein.2. A method as in claim 1 or 2 wherein said at least one predeterminedsingle- or double-stranded nucleic acid is 4 to 1000 nucleotides inlength.
 3. A method for assaying a sample for the presence of aninhibitor of a predetermined nucleic acid binding protein, whichcomprises: a) mixing at least one predetermined single- ordouble-stranded nucleic acid containing at least one label andcontaining a protein binding nucleotide sequence and a predeterminednucleic acid binding protein with a sample which may contain aninhibitor of the binding of said predetermined nucleic acid bindingprotein with said at least one predetermined single- or double-strandednucleic acid, b) incubating the mixture of step a) under conditionswhich allow the binding of said nucleic acid binding protein to said atleast one predetermined single- or double-stranded nucleic acid to forma complex, c) adding a nucleic acid-cleaving enzyme or reagent to themixture of step b), consisting of peptide nucleic acid linkages,phosphorothioate linkages, and methyl phosphonate linkages.
 4. A methodas in claim 1 wherein said label is an electrochemiluminescent label. 5.A method as in claim 4 wherein said electrochemiluminescent label isRuBpy.
 6. A method as in claim 1 or 2 wherein said label is selectedfrom the group consisting of a radioactive moiety, fluorescent moiety,enzyme, chemiluminescent moiety, electrochemiluminescent moiety,bioluminescent moiety, and an optically observable particle.
 7. A methodas in claim 1 wherein said at least one predetermined single- ordouble-stranded nucleic acid sequence has at least one capture moietyattached thereto.
 8. A method as in claim 7 wherein said label is RuBpyand said capture moiety is selected from the group consisting of biotin,avidin, streptavidin, antibody, antigen, lectin, receptor, ligand,hormone, nucleic acid sequence, mimitope, and a nucleic acid basepairing polymer.
 9. A method as in claim 1 or 2 wherein prior to stepa), at least one predetermined single- or double-stranded nucleic acidsequence is contacted with a solid phase.
 10. A method as in claim 1 or2 wherein after step d), the mixture is contacted with a solid phase.11. A method as in claim 1 or 2 wherein said at least one predeterminedsingle- or double-stranded nucleic acid sequence is RNA.
 12. A kitcomprising in one or more containers: a) a nucleic acid having apredetermined protein binding region wherein said nucleic acid has adetectable moiety attached thereto, and wherein said nucleic acid has aplurality of nucleic acid linkages, wherein said linkages preventcleavage of the nucleic acid by a nuclease when a protein is bound tosaid protein binding region, b) a nucleic acid-cleaving enzyme ornucleic acid-cleaving reagent, and c) a solid phase.
 13. A kit as inclaim 12 wherein said nucleic acid has a capture moiety attachedtrereto.
 14. A kit as in claim 12 wherein said detectable moiety is anECL label.
 15. A kit as in claim 14 wherein said ECL label is Ru(byp).16. A kit according to claim 12 wherein said nucleic acid linkages areselected from the group consisting of peptide nucleic acid linkages,phosphorothioate linkages and methyl phosphonate linkages.
 17. A nucleicacid comprising a predetermined protein binding region wherein saidnucleic acid has a detectable moiety attached thereto and wherein saidnucleic acid has a plurality of nucleic acid linkages, wherein saidlinkages prevent cleavage of the nucleic acid by a nuclease when aprotein is bound to said protein binding region.
 18. A nucleic acid asin claim 17 wherein said nucleic acid has a capture moiety attached. 19.A nucleic acid of claim 17 wherein every nucleic acid linkage containedoutside said protein binding region is selected from the groupconsisting of peptide nucleic acid linkages, phosphorothioate linkages,and methyl phosphonate linkages.
 20. A nucleic acid according to claim17 wherein said plurality of nucleic acid linkages are selected from thegroup consisting of peptide nucleic acid linkages, phosphorothioatelinkages, and methyl phosphonate linkages.
 21. A method for assaying asample for the presence of a nucleic acid binding protein, whichcomprises: a) mixing at least one predetermined single- ordouble-stranded nucleic acid containing modified nucleotides that areresistant to nuclease cleavage, at least one label, and containing aprotein binding nucleotide sequence with a sample which may contain anucleic acid binding protein, b) incubating the mixture of step a) underconditions which allow the binding of said nucleic acid binding proteinto said at least one predetermined single- or double-stranded nucleicacid to form a complex, c) adding a nucleic acid-cleaving enzyme orreagent to the mixture of step b), d) incubating the mixture of step c)under conditions which allow the cleavage of said at least onepredetermined single- or double-stranded nucleic acid which has notformed a complex, and e) measuring the amount of said complex by meansthat do not include gel electrophoretic separation to measure saidnucleic acid binding protein.
 22. A method as in claim 21 wherein saidat least one predetermined single- or double-stranded nucleic acidcontains from 1 to 999 modified nucleotides.
 23. A method as in claim 22wherein said modified nucleotides are not contained within said proteinbinding nucleotide sequence.